The present invention relates to methods and reagents for the lysis of erythrocytes in a blood sample. More particularly, the invention relates to the release of hemoglobin by lysis of erythrocytes and the denaturation thereof for the purpose of immunoassay determination of a particular hemoglobin derivative such as hemoglobin A1c.
The determination of the relative amount of hemoglobin that exists in the bloodstream in a particular derivative form generated by in vivo processes is of considerable importance to medical diagnosis. Of particular significance is the measurement of glycated hemoglobins to assess the long term blood glucose control in diabetes mellitus. The glycated form of hemoglobin that perhaps is the most important in diabetes management is the derivative commonly known as hemoglobin A1c. This hemoglobin derivative is produced in vivo by the nonenzymatic reaction of glucose with the hemoglobin protein. Glucose becomes covalently attached in a rearranged form to the amino-terminal valine residue of the beta-chain in native hemoglobin. Normal individuals have between about 3 and about 6 percent of their total hemoglobin in the A1c form, while an uncontrolled diabetic individual could have as much as 3 to 4-fold higher levels of this hemoglobin derivative in the bloodstream.
A variety of methods have been developed for the measurement of glycated hemoglobin. The conventionally used methods are based on diverse techniques such as cation exchange chromatography, electrophoresis, and dye complexation (hydroxyfurfural/thiobarbituric acid). These techniques are well known to be tedious, complex, requiring special technical skills, and suffer from imprecision and nonspecificity. Quite recently, monoclonal antibodies specific for the the glycated N-terminal peptide residue that characterizes hemoglobin A1c have been developed, enabling the detection of this hemoglobin derivative by convenient immunoassay approaches (see U.S. Pat. No. 4,647,654).
The immunoassay determination of hemoglobin A1c using monoclonal antibodies is highly specific and the procedure used is significantly less tedious and complex than the conventional prior art methods. Nevertheless, the optimal hemoglobin A1c immunoassays have been found to require the denaturation of hemoglobin in order to expose the glycated N-terminal peptide epitope for antibody binding (see U.S. Pat. No. 4,658,022). Conditions which have been shown to provide the necessary degree of denaturation of hemoglobin for exposure of the A1c epitope have typically also be sufficient for denaturation of antibody reagents used in the immunoassay detection of the exposed epitope. For example, when using chemical denaturants, it has generally been found necessary to dilute the denatured hemoglobin mixture sufficiently to reduce the concentration of denaturant below that which would significantly interfere with the immunoassay reaction. Such a dilution step is inconvenient and can introduce error to the assay measurement.
An improved denaturant reagent for use in the immunoassay determination of hemoglobin derivatives such as hemoglobin A1c is described in European patent publication 315,864. The blood sample is treated with the combination of a thiocyanate salt and an oxidant to release and denature the hemoglobin in the sample and to convert hemoglobin to its spectrophotometrically detectable met-hemoglobin form. In this way, total hemoglobin can be determined by absorbance measurement of met-hemoglobin and the A1c portion determined and related to total hemoglobin content by immunoassay.
The presence of the met-hemoglobin oxidant during denaturation was found to significantly increase the rate of denaturation of hemoglobin, thereby reducing the overall assay time and improving the reliability of the determination. However, the concentration of thiocyanate salt required for optimal denaturation is sufficiently high that a reduced immunoassay response has been observed. Thus, a dilution step is generally required after denaturation in order to reduce the thiocyanate concentration to a level that does not significantly interfere with the ability of antibody to bind to the exposed A1c epitope.